OUR ENEMY’S ENEMY CAN BE OUR BEST FRIEND

PREPARED BY KAMAL SHRESTHA

BSC MLT

Bacteriophage therapy for antimicrobial resistant and

biofilm forming bacteria

Contents Antibiotic resistance

Common types of resistances Mechanism of resistance Some recent and specific resistant type

Biofilm Formation of biofilm Mechanism of resistance and Quorum sensing

Bacteriophages History of bacteriophages therapyCurrent scenario in bacteriophages therapyAdvantages of bacteriophages therapy Disadvantages of bacteriophages therapyRecent advances in bacteriophages therapySummary and discussion

Antibiotic resistance

Microorganism resistance to an antimicrobial drug that was once able to treat an infection caused by that organism.

“this serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone , of any age , in any places” –WHO

Classification of antimicrobial resistances

Multidrug resistance(MDR) ≥1 agent of ≥3 antimicrobial agents Extensively resistance (XDR)≥ 1 agent in al but ≤2 catergories Pan drug resistance(PDR)non- susceptible to all Antimicrobial agent assign to that bacteria

Mechanism of drug resistance

Drug inactivation/ enzyme modification Alteration of target site Alteration metabolic pathway Reduce drug accumulation Biofilm formation

Biofilm

Biofilm formation is a process whereby microorganisms irreversibly attach to and grow on a surface and produce extracellular polymers that facilitate attachment and matrix formation, resulting in an alteration in the phenotype of the organisms with respect to growth rate and gene transcription.

Biofilms are resistant to killing by antibiotics at concentrations that are 10-1000 times greater than concentrations needed to kill free-living or “planktonic”

often lead to life-threatening systemic infections and device failure

Steps of biofilm formation

Attachment of cell to any surface

Cell attach to surface irreversibly (secretion of extracellular polymeric substance)

Cells adsorbed on surfaces replicatean grow on micro colonies

Community grows into a three dimensional and formation of mature biofilm

Some cell detach from the region of biofilm

Mechanism of resistance

1. Restricted penetration of antibiotics2. Nutrient limitation, altered

microenvironment3. Adaptive response 4. Quorum sensing5. Genetic alteration to persister cell

Bacteriophages

virus that infects and replicates with in bacterium They do so by inserting their genetic material inside a bacteria composed of protein capsule containing either DNA or RNA as their genomeThey are the most common and diverse entities in biosphere and thrives where there is a high bacterial populationThe phages are terraforming the planet. Every second day the phages destroy approximately 50 percent of the Earth's bacterial population.

Different types of phages

History and taxonomy

Ernest Hankin - 1896Frederick Towrt - 1915Felix d’Herelle – 1917

Taxonomy(ICTV)Order Caudovirales Ligamenvirales not assign Myoviridae lipoyhrixviridae Ampullaviridae T4 phage Mu Acidious Bicaudaviridae, etc. filamentous virus Siphoviridae Rudiviridae λ phage, T5 sufolobus islandicus virus Podoviridae T7 T3 phages

Life cycle

From where we can get phages

Obtain a fresh culture of bacteria and make a an overnight suspension with suitable broth

Add certain volume of sample(5ml) + 0.5ml of overnight suspensions + 0.5ml of 10x brothand

incubate

Centrifuge the incubated suspension for 10 minsat 2500rpm and take the supernatant ins separate tube

and filter by using <0.4micron filter

Take small volume from filtrate(10µl) and add to the lawn culture of bacteria

Harvest the phage by using loop and add into another suspension of same bacteria and store in refrigerator

Bacteriophage therapy

Use of bacteriophages for the therapeutic purpose in bacterial infection

This method is still not approved in all countries except Georgia

This method is still being testing for the treatment of antimicrobial resistance and biofilm forming bacteria

Why phage therapy

History of phages therapy 1919- 111940 , golden age for phage therapy 1919 Felix d’Herelle extensively studies about the phenomenon of

bacteriophage and also used in human suffering from dysentery at Hôpital des Enfants-Malades in Paris.

1921 – 1st reported case of bacteriophage used to treatment of bacterial infection in human by Richard Brugnoghe and Joseph Maisin

D'Herelle's commercial laboratory in Paris produced at least five phage preparations against various bacterial infections.

1940 discovery of antibiotic halted the research on bacteriophage except in east Europe

Eliava Institute of Bacteriophage, Microbiology, and Virology (EIBMV) of the Georgian Academy of Sciences, Tbilisi, Georgia, and the Hirszfeld Institute of Immunology and Experimental Therapy (HIIET) of the Polish Academy of Sciences, Wroclaw, Poland were among the institute which done most of its work in bacteriophage therapy

Bacteriophage in animal trialsSmith et al – 1982, successful use of phage to

experimental E. coli infection in mice. Phages treatment reduce the no. of bacteria by many fold in different animals. Rekindled the concept of bacteriophage therapy in West

Soothil et al- (1988-1994), successfully treated the experimental disease caused by Pseudomonas and Acinetobacter in mice and guinea pig and suggested that it might be efficacious in preventing infection of skin graft and burn patient.

Bogovazova et al- 1191, reported that phages are efficacious and non-toxic (no gross and histological changes) even after the use of 3,500 fold higher than that use in humans , in mice and guinea pigs.

Bacteriophage in human trialsPolish paper:-1983-1985 Slopek et al published the SIX paper on effectiveness of the bacteriophage against

infection caused by MDR and found that bacteriophages are 75-100% effective(>94%) Another study also reported the effectiveness of bacteriophages therapy in meningitisSoviet paper:- 1963-1964 In Georgia the effectiveness of bacteriophage to treat dysentery was determined and

found that overall 3.8 fold lower incidence of dysentery in children given anti-shigella bacteriophage orally.

In many other study yielded the similar result but main drawback of soviet studies was it lacks the information required for the determination efficacy of bacteriophage therapy

Other studies Zhukov-Verezhnikov et al -1978, compared the effectiveness of

specially adapted bacteriophage to commercially available bacteriophage and found that adapted bacteriophages are 5-6 fold more effective.

Meladze et al- 1982, compared the effective ness of bacteriophage to antibiotic and found to superior to antibiotic with lesser side effect

Advantages of phage therapy highly specificVery effectiveHarmless/very low side effectPhage are intelligent drug Bacteria fully resistant to phage hasn’t yet discovered Easy availability Low cost for the preparation Effective against most resistant bacteriaEffective even to mature form of biofilm Phage can be genetically modified Individual component can be used to treat patientPhages mutate at a higher rate than bacteria and are able

to respond fast to possible phage- resistant bacteria.

Disadvantages/problems of phage therapy

Efficacy of phages in human hasn’t been full determined internationally

High specificity has hindered its effectiveness to many bacteria

Purity of bacteriophages suspensionBacteria resistant to phagesLarge size of phages Intracellular pathogen Clearance of phages by reticulo-endothelial systemFormation of antibodies against bacteriophagesRelease of cellular toxins during cell lysisCan carry harmful gene to bacteria(lysogeny) Difficulty in administration

Prerequisites of bacteriophage therapy

Phage therapy should not be attempted before the biology of the therapeutic phage is well understood.

Phage preparations should meet all the safety requirements

Phage preparations should contain infective phage particles,

The phage receptor should be known.The efficacy of phage therapy should be

tested in an animal model.

Recent advances to tackle the problems

Cocktail of bacteriophages:-Broader phages Ellen et al , 1998 has succeed to isolates a

different bacteriopages with broader host range such as SN-1, SN-2, SN-T, and SN-X, AB1157, BHR3, BHR4, and BHR5 which can infected staphylococcus natans Pseudomonas aeruginosa and escherichia coli.

Merril et al- 1996 succeed to produce a mutant bacteriophage by serially passaging phages through animal which can stay to circulation more longer period of time

Drug- delivery technologies Kim et al- 2008, conjugated the bacteriophage to polyethylene glycol(PEG) and found that this conjugation has the increase the sustainability of bacteriophages in circulation and also decrease the production of Th1 and interleukin factor showing decrease immune response

Genomic modification of bacteriophage:-

Non-lytic/non-replicative Hagens et al– 2003, genetically modified filamentous bacteriophages

by replacing the export protein gene with restriction endonuclease but not holins which are lethal to bacteria but do not induce lysis of bacteria.

Lu and colins- In 2007, show that phage can be genetically modified to

disrupt the barrier like biofilm, they inserted dispersin-B( glycoside hydrolase known to degrade biofilm in Escherichia bacteriophage T7.

In 2009 ,they genetically modified M13mp18 phages that overexpress the lecA3 gene which decrease the SOS response(DNA repair) on the presence of quinolones and found to be effective in combating resistance bacteria.

Bacteriophage product

Enzybiotic – Nelson et alLysin:- endolysins or murein hydrolases are the hydrolytic

enzymes produced by bacteriophages in order to cleave the host’s cell wall during the final stage of life cycle.

Recombinant enzymes acting on cell wall can be uses for therapeutic purpose rather than a whole bacteriophage

Lysin effectiveness to eliminate the infection has been shown by many study

e.g. Nelson et al-2001 use C 1 bacteriophage lysin to treat a experimental infection with streptococci of upper respiratory tract in mice. And shows the high rate of activity

List showing specific bacteriophage ant their lysin for different bacteria

continue

Protein antibiotics

Some small phages do not have the genes for holin or lysin proteins.

Instead, they produce a protein that inhibits a step in murein monomer synthesis. Their inhibitory gene products are known as “protein antibiotics”

the E protein of the single-stranded (ss)-DNA bacteriophage φX174 (Microviridae), (ii) the L protein of the ss-RNA bacteriophage MS2 (Leviviridae), and (iii) the A2 protein of the ss-RNA bacteriophage Qβ (Alloleviviridae) are some of protein that can induce cell lysis in similar manner to that of penicillin.

A Trojan Horse Approach

Killing of Mycobacterium avium and Mycobacterium tuberculosis by a Mycobacteriophages delivered by a Non-virulent Mycobaterium(M smegmatis)

Infected M smegmatis with a TM4 phages(a broader ranges phage that infect from fast growing mycobacteria to slow growing Mycobacteria)

Result shows that its able to kill tha micobacteria in free living state as well as that are inside the macrophage

Bacteriophages vs antibiotics

Use of phages in other industries

In food industry Bacteriophage bioprocessing , a means of

reducing bacteria from food product by using bacteriophages

This non-thermal intervention has been demonstrated to control the growth of many bacteria e.g. campylobacter and salmonella on chicken listeria in meat etc.

In agriculture and fisheries

Phage typing

Is also known as the use of sensitivity pattern to specific phages to precisely identifying the microbial strains

It implies the use of a set of bacteriophages provided by international agency for the typing of certain species of bacteria for epidemiological purpose

Phage for the detection of bacteria

Wild phage detection system:-

reporter bacteriophages :-

modified phages used as a reporting gene carrier, introducing a gene of interest into the host bacteria upon infection

Eg luciferase expressing gene(lux and luc) galactosidase(lacZ), bacterial ice nucleation(inaW) , green fluorescent protein(gfp) expressing gene

Phages receptor binding proteins

This are unique protein located on the tail fibers , which binding to the host receptors induce the translocation of genetic materials

Bacteria can be detected by using these protein like a antibody which binds to the specific bacterial.

Offers better stability against many environmental factors such as pH temperature and different enzymes(proteses)

Binding affinity can be tailored to the requirementSingh et al. demonstrated the use of cysteine-tagged P22

phage RBPs on gold surface for captureand detection of Salmonella enterica serovar

Typhimurium.

Conclusion

Multidrug resistant bacteria have opened a second window for phages therapy

Modern innovation combined with careful scientific methodology, can enhance mankind’s ability to make it work this time around

Phage therapy can stand alone therapy for infectious that are fully resistant

It will also then be able to serve as a co-therapeutic agents for infections that are still susceptible to antibiotic by helping to prevent the emergence of bacterial mutant against either agent

references

Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G., ... & Monnet, D. L. (2012). Multidrug‐resistant, extensively drug‐resistant and pandrug‐resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection, 18(3), 268-281.

Sulakvelidze, A., Alavidze, Z., & Morris, J. G. (2001). Bacteriophage therapy. Antimicrobial agents and chemotherapy, 45(3), 649-659.

Skurnik, M., & Strauch, E. (2006). Phage therapy: facts and fiction. International Journal of Medical Microbiology, 296(1), 5-14.

Lu, T. K., & Koeris, M. S. (2011). The next generation of bacteriophage therapy. Current opinion in microbiology, 14(5), 524-531.

Harper, D. R., Parracho, H. M., Walker, J., Sharp, R., Hughes, G., Werthén, M., ... & Morales, S. (2014). Bacteriophages and biofilms. Antibiotics, 3(3), 270-284.

Merril, C. R., Biswas, B., Carlton, R., Jensen, N. C., Creed, G. J., Zullo, S., & Adhya, S. (1996). Long-circulating bacteriophage as antibacterial agents. Proceedings of the National Academy of Sciences, 93(8), 3188-3192.

Lu, T. K., & Collins, J. J. (2009, April). Engineering synthetic bacteriophage to combat antibiotic-resistant bacteria. In Bioengineering Conference, 2009 IEEE 35th Annual Northeast (pp. 1-2). IEEE.

Continue

Heo, Y. J., Lee, Y. R., Jung, H. H., Lee, J., Ko, G., & Cho, Y. H. (2009). Antibacterial efficacy of phages against Pseudomonas aeruginosa infections in mice and Drosophila melanogaster. Antimicrobial agents and chemotherapy, 53(6), 2469-2474.

Borysowski, J., & Górski, A. (2010). Enzybiotics and their potential applications in medicine (pp. 1-26). Wiley, New York.

Lukacik, P., Barnard, T. J., Keller, P. W., Chaturvedi, K. S., Seddiki, N., Fairman, J. W., ... & Buchanan, S. K. (2012). Structural engineering of a phage lysin that targets gram-negative pathogens. Proceedings of the National Academy of Sciences, 109(25), 9857-9862.

Ghannad, M. S., & Mohammadi, A. (2012). Bacteriophage: time to re-evaluate the potential of phage therapy as a promising agent to control multidrug-resistant bacteria. Iranian journal of basic medical sciences, 15(2), 693.

Bernhardt, T. G., Wang, N., Struck, D. K., & Young, R. (2002). Breaking free:“protein antibiotics” and phage lysis. Research in microbiology, 153(8),493-501.

Broxmeyer, L., Sosnowska, D., Miltner, E., Chacón, O., Wagner, D., McGarvey, J., ... & Bermudez, L. E. (2002). Killing of Mycobacterium avium and Mycobacterium tuberculosis by a mycobacteriophage delivered by a nonvirulent mycobacterium: a model for phage therapy of intracellular bacterial pathogens. Journal of Infectious Diseases, 186(8), 1155-1160.